Neuroblastoma is the most common solid tumor of infancy and the most common extracranial solid tumor of childhood, accounting for more than 7% of childhood cancers and 15% of cancer-related childhood deaths (Maris et al., Lancet, 2007, 369(9579):2106-20; Park et al., Hematol Oncol Clin North Am, 2010, 24(1):65-86). Neuroblastoma arises from the neural crest cell precursors of the sympathetic nervous system that fail to differentiate (Park et al., Hematol Oncol Clin North Am, 2010, 24(1):65-86; Brodeur, Nat Rev Cancer, 2003, 3(3):203-16)—this provides the basis for differentiation therapy, an approach to induce malignant cells to differentiate into mature cells, thereby leading to cell growth arrest and apoptosis (Park et al., Hematol Oncol Clin North Am, 2010, 24(1):65-86; Reynolds, Curr Oncol Rep 2000, 2(6):511-18; Cruz and Matushansky, Oncotarget, 2012, 3(5):559-67; Nowak et al., Blood, 2009, 113(16):3655-65). However, only a limited number of differentiation agents have been successfully used to treat neuroblastoma. The differentiation agent 13-cis-retinoic acid (RA) is currently the standard of care for post-remission maintenance therapy in high-risk neuroblastoma (Park et al., Hematol Oncol Clin North Am, 2010, 24(1):65-86). Although such treatment has resulted in a significant increase in patient survival, more than 50% of the treated patients still develop recurrence (Matthay et al., J. Clin Oncol, 2009, 27(7):1007-13; Matthay et al., N Engl J Med, 1999, 341(16):1165-73). Such poor outcomes demand the development of new differentiation agents. Unfortunately, the mechanisms that result in the loss of differentiation ability of neuroblastoma cells are poorly understood, which poses an obstacle to such development. Therefore, identifying additional differentiation agents largely relies on the discovery of new targetable biological molecules that play critical roles in neuroblastoma differentiation.
High-throughput screening approaches significantly facilitate the discovery of novel anti-cancer drugs and drug targets. More recently, high-content screens (HCSs) based on automated cell imaging have been developed. However, current HCSs generally either use genetic engineered cell lines expressing fluorescent signals or involve staining of fixed cells (Conrad and Gerlich, J Cell Biol, 2010, 188(4):453-61; Jan et al., ACS Nano, 2008, 2(5):928-38), which are generally time-consuming and consequently limit their broad applications to drug discoveries.
miRNAs are endogenously expressed small RNAs that play a critical role in tumorigenesis (Shenouda and Alahari, Cancer Metastasis Rev, 2009, 28(3-4):369-78; Li et al., Aaps J, 2010, 12(3):309-17; Kinoshita et al., Oncotarget, 2012, 3(11):1386-1400; Kopp and Roidl, Oncotarget 2013; Bier et al., Oncotarget, 2013, 4(5):665-76). The therapeutic potential of either exogenously increasing cellular miRNAs levels with synthetic miRNA mimics, or inactivating endogenous miRNAs with synthetic miRNA inhibitors has been demonstrated in previous studies (Kota et al., Cell, 2009, 137(6):1005-17; Krutzfeldt et al., Nature, 2005, 438(7068):685-89; Trang et al., Mol Ther, 2011, 19(6):1116-22). The role of miRNAs in neuroblastoma differentiation and tumorigenesis has been implicated (Lin et al., Cancer Res, 2010, 70(20):7841-50; Chang et al., Nat Genet, 2008, 40(1):43-50; Wei et al., Oncogene, 2008, 27(39):5204-13; Tivnan et al., BMC Cancer, 2011, 11:33; Makeyev et al., Mol Cell, 2007, 27(3):435-48; Annibali et al., PLoS One, 2012, 7(7):e40269; Le et al., Mol Cell Biol, 2009, 29(19):5290-305; Foley et al., Cell Death Differ, 2011, 18(7):1089-98; Swarbrick et al., Nat Med, 2010, 16(10):1134-40), which suggests the potential of developing novel miRNA-targeting approaches to neuroblastoma differentiation therapy (Mishra and Merlino, J Clin Invest, 2009, 119(8):2119-23), and warrants a comprehensive understanding of the involvement of miRNAs in neuroblastoma cell differentiation. However, there has been no concerted effort to comprehensively investigate the functions of the miRNA species in neuroblastoma differentiation.